Evaluation of podocyte lesion in patients with diabetic nephropathy: Wilms’ tumor-1 protein used as a podocyte marker

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Abstract

Introduction

The reduction of podocyte number and density per glomerulus has been linked to the development of proteinuria and the progression of disease in patients with diabetic nephropathy (DN). However, it has been recognized that measurement of podocyte number by light microscope is quite difficult because of the complexity of both podocyte and glomerular structure, which is not suitable for clinical research. In our research institute, we used WT1 as podocyte marker to evaluate the podocyte lesion.

Methods

In our experiment, we selected the C-terminal antibody of WT1 to stain the nuclei and the N-terminal antibody of WT1 to stain the cytoplasma of podocytes. Forty patients were enrolled with type 2 diabetes and proven to have DN by renal biopsy analysis. DN patients were classified into three groups based on the degree of proteinuria: microalbuminuria (n = 10, 30–300 mg/24 h), overt proteinuria (n = 15, 0.5–3.5 g/24 h), and heavy proteinuria (n = 15, >3.5 g/24 h).

Results

The results demonstrated that the podocyte number was markedly decreased in patients with DN (30–51% reduction). There was a significant negative correlation between the proteinuria and both podocyte density and number. The cover area density of podocyte cytoplasma in glomerulus was also significantly decreased in all DN patients (39–80% reduction). A significant inverse correlation was observed between the cover area density and the degree of proteinuria. The correlation coefficient (r = −0.85) was much higher than that between proteinuria and podocyte density (r = −0.56) or podocyte number (r = −0.36).

Conclusion

In conclusion, podocyte damage occurred in patients with DN, even in the early stage and became more dramatic during the course of proteinuria progression. WT1 staining, using the polyclonal antibody to stain the nuclei and monoclonal antibody to stain the cytoplasma of podocytes together, is a valuable alternative technique in the study of podocyte injury.

Introduction

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease and is clinically characterized by proteinuria and progressive renal insufficiency. Expansion of the mesangial area and thickening of the glomerular basement membrane (GBM) are two characteristic histological features of DN [1]. The molecular mechanisms of how hyperglycemia induces extracellular matrix synthesis and thickening of the GBM have been intensively studied [2], [3], [4]. Numerous cytokines, such as transforming growth factor-β, connective tissue growth factor, angiotensin II are considered to be implicated in these pathophysiological changes [5], [6], [7]. However, the magnitude of these lesions does not completely explain the development of abnormal albuminuria in DN patients. For example, despite persistent microalbuminuria or proteinuria some patients with type 2 diabetes have normal or near normal renal structure [8], [9]. In vivo experiments also find no evidence that changes in mesangial area or the structure of GBM play a pivotal role in microalbuminuria and early proteinuria in DN [4], [10].

Recently, increasing evidence implicates the podocyte as an important player in the initiation and development of proteinuria in DN. Steffes et al. found that podocytes were fewer in number in type 1 diabetic patients of all ages, with reduced podocyte numbers even in diabetes of short duration [11]. In a European cohort of type 2 diabetic patients, Vestra et al. found a significant reduction in the numerical density of podocytes in normoalbuminuric patients that further decreased in the proteinuric patients [12]. In a Japanese study, podocytes were found in the urine in 53% of type 2 diabetic patients with microalbuminuria and in 80% with macroalbuminuria [13]. These studies provide convincing evidence that podocyte lesion exists even before the development of proteinuria.

We noted that these studies observed the podocyte lesion by counting the number of podocytes in the glomeruli with light microscope [11], [12], [14], [15], [16], [17]. However, it has been recognized that measurement of podocyte number by light microscope is quite difficult because of the complexity of both podocyte and glomerular structure [18], which is not suitable for clinical research. Thus it is necessary to discover a convenient marker to study the podocyte in patients with renal diseases.

Wilms’ tumor-1 (WT1) is a zinc-finger containing transcription factor involved in the nephrogenesis and podocyte differentiation. In the mature glomerulus, the expression of WT1 is restricted to podocyte [19]. A few reports have used WT1 C-terminal polyclonal antibodies (WT1-C) to stain renal specimens and found that WT1-C is exclusively expressed in the podocyte nuclei, whereas nuclei of mesangial cells and endothelial cells are negative [20], [21], [22]. By using this WT1-C antibody, one can identify podocyte accurately and conveniently, so we selected this antibody to count podocytes in our studies.

Indeed, besides the decrease in the number, the morphological changes of podocytes, related to the enlargement of glomerular volume and/or the redistribution of podocyte associated proteins and other factors, are also the key elements in the progression of DN, especially in the early stage of the disease [12]. A few studies had reported that the WT1 N-terminal monoclonal antibody (WT1-N, Clone 6F-H2) produced a cytoplasmic staining pattern in podocytes that differed with the studies using WT1-C antibody [23], [24], [25], [26]. It was an exciting finding because it gave us the opportunity to observe the podocyte cytoplasma which can closely reflect the structure of podocytes.

Thus, in the present study, we selected WT1-C antibody to stain the nuclei and WT1-N antibody to stain the cytoplasma of podocytes. We detected the number and density of podocytes and estimated the distribution of podocyte cytoplasma in patients with DN. Our aim was to find out a convenient marker to study the podocyte in patients with renal diseases.

Section snippets

Patients

Forty patients with type 2 diabetes and DN proven by renal biopsy were enrolled in this study. Type 2 diabetes was diagnosed according to American Diabetes Association criteria [27]. Patients were excluded when they had non-diabetic renal disease. DN patients were classified into three groups based on the degree of proteinuria: (1) microalbuminuria (n = 10) defined as urine protein negative by routine method, while urinary albumin excretion rate (AER) within the extent of 30–300 mg/24 h as measured

Common clinical features of patients

Forty patients with type 2 diabetes were included in this study. Patients with heavy proteinuria had longer duration of the disease and higher serum creatinine than other DN patients. Fasting blood glucose and HbA1c were similar in these three groups. Main clinical and laboratory characteristics are given in Table 1.

Podocyte WT1 expression in patients with DN

In all the specimens that observed, WT1-C antibody was mainly localized in the podocyte nuclei, whereas nuclei of mesangial cells and endothelial cells were WT-1 negative. The

Discussion

Recently, podocyte had become the focus of research into the mechanisms of progression of nephropathies. In DN, many studies had observed that the changes in podocyte number and density were related to proteinuria and damage to the podocyte was instrumental to the progression of kidney diseases [11], [12], [14], [15], [16], [17]. However, in their studies most authors measured podocyte number by light microscope, which was difficult and not suitable for clinical research.

In the present study,

Conflicts of interest

There are no conflicts of interest.

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